Obesity and the metabolic syndrome are global health threats. Intracellular triacylglycerol (TG) accumulation is strongly associated with insulin resistance and metabolic complications of obesity. However, the mechanisms underlying TG accumulation and its relationship to lipid-induced toxic metabolic effects (lipotoxicity) remain unclear. Emerging evidence suggests that non-TG lipid metabolites (i.e. diacylglycerols, ceramides, fatty-acyl CoAs, etc.) rather than TGs themselves are pathogenic for these disorders, indicating that interventions designed to enhance TG storage and/or reduce production of toxic lipid metabolites may protect against insulin resistance and lipotoxic metabolic disease. Adipose triglyceride lipase (ATGL) has recently been identified as the rate-limiting enzyme mediating TG hydrolysis, and is therefore a critical determinant of storage/production of toxic as well as essential lipid metabolites. Nevertheless, the role of ATGL in these processes, particularly in non-adipose tissues where ATGL is also expressed, remains poorly understood. Our preliminary data suggest that i) ATGL is expressed, highly regulated, and functionally relevant in adipose and non-adipose tissues, and ii) modulation of ATGL-mediated TG hydrolysis influences glucose homeostasis/ insulin action in vitro and in vivo in a tissue/cell type-specific manner. Understanding the relative contribution of ATGL in individual tissues to overall metabolic homeostasis in vivo as well as the mechanisms by which ATGL exerts these effects are essential for determining whether TG metabolism can be therapeutically modulated to protect against lipotoxic metabolic disease. The CENTRAL AIM of this proposal is to determine the tissue- specific contribution of ATGL-mediated TG hydrolysis in metabolically relevant tissues (adipose tissue and skeletal muscle) to lipotoxicity and the metabolic syndrome in vivo using genetically-engineered mice as model systems. Our OVERALL HYPOTHESIS is that dysregulation of ATGL-mediated TG hydrolysis, by modulating intracellular lipid homeostasis, is a major determinant of both tissue-specific and systemic glucose homeostasis/insulin action. To test this hypothesis, we will evaluate local and systemic lipid homeostasis and glucose homeostasis/insulin action in mice with tissue-specific modulation of ATGL in adipose tissue (AIM 1) and skeletal muscle (AIM 2). We will further define the molecular mechanisms by which modulation of ATGL alters lipid homeostasis and glucose homeostasis/insulin action by evaluating specific mechanisms that contribute to lipotoxicity (i.e. endoplasmic reticulum stress, mitochondrial dysfunction, inflammation, etc.). These studies will promote the understanding of ATGL in tissue-specific and systemic metabolism, thereby providing important insights into the pathogenesis and treatment of obesity and related metabolic disorders. This is the first R01 application of an early stage investigator.